Compositions and methods for treatment of microbial infections

ABSTRACT

The present invention relates to methods and compositions for treatment of microbial infections and for the enhancement of resistance to infection. The invention comprises administration of an effective amount of bacterial lysate compositions for the treatment of pathological conditions of microbial infections. The present invention can also be used to enhance the immune system to prevent infections by the administration of an effective amount of the compositions.

RELATED APPLICATION

[0001] This application claims benefit of U.S. Provisional ApplicationNo. 60/356,483 filed Feb. 13, 2002, which is incorporated herein in itsentirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the treatment ofmicrobial infections. More specifically, the invention relates tomethods and compositions for the enhancement of an immune response in ahuman or animal to microbial infections.

BACKGROUND OF THE INVENTION

[0003] The idea of using phages for treatment of infectious diseases wasinitially proposed by d'Herelle in 1917. In the 1920s, the discovery ofbacteriophages was thought to be the answer for treatment of bacterialinfections. Bacteriophages invade and destroy bacteria and appeared tobe the selective therapeutic bullet that would knock out bacteriainvading an animal or plant host. Unfortunately, the phages were not aseffective in killing bacteria in host organisms as they were in killingbacteria in vitro. The development of antibiotics in the 1940s as thepreferred treatment for bacterial infections led to a decline inresearch into use of bacteriophages for treatments of infectiousdiseases.

[0004] In a few countries, notably in Eastern Europe and India, researchcontinued in the use of bacteriophages and bacterial lysates fortreatment of infectious diseases and enhancement of immune responses.Though much of that research is still not accepted widely in otherindustrialized countries, a renewed interest in this research is growingas a result of the occurrence of antibiotic resistant bacteria. Manyinfectious diseases that were once easily treatable with antibiotics arenow a serious health threat because the bacteria are resistant to most,if not all, antibiotics. Medical science is searching for treatmentsthat can respond to these resistant bacteria in a way that does not leadto even more resistant infectious strains.

[0005] The U.S. Centers for Disease Control and Prevention estimatesthat 20% to 50% of prescribed antibiotics are unnecessary. The overuseof antibiotics in medicine and agriculture has greatly fostered thedevelopment of resistant strains of bacteria. In a microenvironmentbombarded with antibiotics, the few bacteria that can resist the drugsproliferate. Bacteria are becoming increasingly resistant to what werepreviously considered “last resort” antibiotics.

[0006] Bacteriophage therapy has not gained much attention or acceptancein most industrialized countries, in part due to the reliance onpharmaceuticals, such as antibiotic treatment for bacterial infections.With the advent of increased bacterial resistance, bacteriophage therapyhas come under renewed scrutiny as a possible alternative topharmaceutical treatments of bacterial infections. What is needed arecompositions and methods for treatment and prevention of microbialinfections that do not rely on pharmaceutical antibiotic therapies. Suchmethods and compositions should be capable of treating or preventinginfections in organisms, including humans, animals and plants.

SUMMARY OF THE INVENTION

[0007] The present invention is directed towards compositions andmethods of treatment and prevention of microbial infections. Preferredmethods include administration of compositions comprising bacteriallysates. The present invention contemplates use of any bacterium thathas stable reproduction and does not produce a toxin or have deleteriouseffects either on the bacteriophage or on the human or animal receivingthe resulting lysate. Preferred bacterial lysates include, but are notlimited to, those derived from strains of Staphylococcus aureus (S.aureus), Klebsiella pneumoniae, (K. pneumoniae) and Pseudomonasaeruginosa (P. aeruginosa). Most preferred bacterial strains comprisebacterial strains deposited at the Czech Collection of Microorganisms(CCM) having accession numbers, CCM 4992, CCM 4993, CCM 4994, CCM 4995,CCM 4996, CCM 4997, CCM 4998. Most preferred bacteriophages comprisebacteriophages deposited at the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSM) and having accession numbers, 14614, 14615,and 14616.

[0008] The present invention comprises a composition, comprising abacterial lysate derived from the infection of at least one bacterialstrain of S. aureus, K. pneumoniae, or P. aeruginosa with abacteriophage. The composition of the present invention furthercomprises a bacterial lysate derived from one or more S. aureusbacterial strains deposited with the CCM under accession numbers CCM4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM 4997, and CCM 4998.The present invention also comprises a bacterial lysate derived frombacteriophages deposited with the DSM having accession numbers DSM14614, DSM 14615, and DSM 14616. The present invention additionallycomprises S. aureus bacterial isolates deposited with the CCM underaccession numbers CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM4997, and CCM 4998. The present invention also comprises bacteriophages,deposited with the DSM under accession number DSM 14614, DSM 14615, andDSM 14616.

[0009] The bacterial lysates of the present invention may be used inmethods of treating or preventing pathological conditions of microbialinfections in humans or animals. The compositions and inventions of thepresent invention may further comprise a suitable pharmaceuticalexcipient. The pathological conditions of microbial infections which canbe treated or prevented by the present invention include, but are notlimited to, conditions such as chronic upper respiratory disease, woundinfection, osteomyelitis, endocarditis, skin polymicrobial infections,bronchial asthma, chronic sinusitis, cystic fibrosis or acne vulgaris.

[0010] The compositions of the present invention may be used as vaccinecompositions as well as in the treatment specific microbial infections.The compositions of the present invention may consist of one or morebacterial lysates formed by infecting specific strains of bacteria withbacteriophages. The compositions can be used in methods for thetreatment and prevention of local or systemic bacterial infectionsincluding, but not limited to, chronic or recurrent respiratory or earinfections, post operational infections, bacterial pneumonia infections,sepsis, skin infections, wound infection, osteomyelitis, allergies,asthma, sinusitis, and acne vulgaris. Additionally, the compositions ofthe present invention may be used in methods for immune stimulation.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention comprises compositions and methods for thetreatment of microbial infections comprising administering an effectiveamount of one or more bacterial lysates or mixtures thereof. Theimmunogenic compositions of the present invention may further compriseat least one or more immunogenic or immunostimulating materials orformulations for regulating or affecting microbial distribution in anorganism.

[0012] The present invention also comprises compositions and methods ofvaccination against microbial infections comprising administeringcompositions comprising one or more bacterial lysates or mixturesthereof and a pharmaceutically acceptable carrier. The lysates may beused individually or in combination. The vaccines of the presentinvention are used to immunize animals, including humans, againstbacterial diseases by administering to the human or animal an effectiveimmunizing amount of the bacterial lysate.

[0013] The compositions of the present invention comprise bacteriallysates produced by infecting selected bacteria with selectedbacteriophages. The present invention contemplates use of anybacteriophage that stably reproduces in a selected bacterial strain anddoes not result in the production of a significant amount of a toxin oranother component that has deleterious effects either on the hostbacteria or in the resulting lysate that is deleterious to the human oranimal receiving the lysate. Determination and selection of suchbacteriophages are performed using techniques known to microbiologistsand include testing for long term stability in a general host strain,long term persistence of virulence, stability and reproducibility oflysis and reproducibility of resultant lysates. The host strain is astrain which is extremely sensitive to the lytic properties of thebacteriophage. It is selected from the strains sensitive to theparticular bacteriophage.

[0014] The compositions of the present invention further comprisebacterial lysates derived from selected bacteria. The present inventioncontemplates use of any bacterium that has stable reproduction and doesnot produce a toxin that has deleterious effects either on thebacteriophage or in the lysate recipient. Determination and selection ofsuch bacteria is performed using techniques known to those skilled inthe art and includes testing for the absence of toxins particularly:alpha toxin, beta toxin, delta toxin, gamma toxin, enterotoxins A, B, C,and D, Toxic Shock Syndrome Toxin (TSST), exfoliatins A and B,leukocidin, fatty acid modifying enzyme, and hemolysins. Testing alsoincludes tests for stability and reproducibility of lysates generated bythe phage-induced lysis, long-term sensitivity of the bacterial strains,and continued susceptibility of the bacteria to lysis by phages withoutthe appearance of phage-resistant colonies.

[0015] Selection of a lysate composition is determined by the methods ofuse for a particular lysate composition. For example, if the desired useis to provide immunity for staphylococcal infections, one or morestrains of staphylococcal bacteria are used as the bacterial hostorganisms. In this same example, one or more bacteriophages that arespecific for staphylococcal bacteria, or are at least capable of havinga productive infection in staphylococcal bacteria, are used to createthe staphylococcal lysate. Alternatively, one bacterial strain may begrown to produce a bacterial culture or bacterial broth and thenseparate aliquots of the bacterial culture are each infected with adifferent bacteriophage to create individual lysates. These individuallysates may be used individually or combined to form compositions. Inanother embodiment, different bacterial strains are grown and then eachis infected with the same bacteriophage to yield lysate compositionsthat can be used individually or combined to form compositions. Anotherembodiment of the present invention contemplates the use of differentbacterial strains that are each infected with different bacteriophagesand the resulting lysates are used individually or in combination toform compositions.

[0016] The compositions of the present invention preferably comprisecompositions comprising lysates from at least one bacterial strain, morepreferably, two or more bacterial strains. A preferred compositioncomprises lysates from one of the following bacteria: Staphylococcusaureus (S. aureus), Klebsiella pneumoniae (K. oneumoniae), andPseudomonas aeruginosa (P. aeruginosa). Preferred compositions compriselysates derived from bacterial strains of S. aureus deposited with theCzech Collection of Microorganisms (CCM) on Oct. 11, 2001 and havingaccession nos. CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM4997 and CCM 4998. Preferred compositions additionally comprise thebacterial strains deposited with the CCM on Oct. 11, 2001 and havingaccession numbers CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM4997 and CCM 4998

[0017] The methods of the present invention comprise the use ofbacteriophages to yield the desired bacteriophage lysates. A morepreferred method comprises lysates produced by bacteriophages for S.aureus, K. pneumoniae, and P. aeruginosa. Most preferred methodscomprise lysates derived from bacteriophages deposited on Nov. 19, 2001with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSM) and having accession numbers 14614, 14615, and 14616. Preferredcompositions additionally comprise the bacteriophages deposited on Nov.19, 2001 with the Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH (DSM) and having accession numbers 14614, 14615, and 14616. Thebacteriophages can be used alone or in combination to lyse one or moredifferent bacterial strains.

[0018] Generally, the methods of the present invention comprise growingone or more desired bacterial strains to a desired concentration. Forexample, ranges of preferred concentrations of bacterial cultures arefrom 1×10⁶ to 1×10¹² cells/ml, preferably 1×10⁷ cells/ml, 1×10⁸cells/ml, 1×10⁹ cells/ml, 1×10¹⁰ cells/ml, 1×10¹¹ cells/ml. At theselected time, one or more bacteriophages, at an effectiveconcentration, are added to the bacterial culture. For example, rangesof preferred concentrations of bacterial phage cultures are from 1×10⁸to 1×10¹², preferably, 1×10⁹, 1×10¹⁰, 1×10¹¹. Infection and lysis by thebacteriophages is allowed to proceed. Incubation of the bacteriophage orphages with the bacterial culture can be from 3 to 48 hours. Theresulting bacterial lysate is then filter sterilized using filtershaving a pore size from <0.1 μm to 0.45 μm.

[0019] Both the material retained on the filter, the rententate, and theflow through material, the filtrate, are kept. It is preferred to usethe filtrate for compositions in methods of treatment and immunestimulation. In general, the filtrate comprises cellular materials suchas components of cell walls, cellular membranes, proteins, ribosomalfractions, glycoproteins, DNA, RNA, and the like. The rententate fromultrafiltration using filters with a pore size of <0.1 micrometerscomprises larger cellular materials and unlysed cells and bacteriophageswhich may also be used in the compositions and methods of the presentinvention.

[0020] The use of bacteriophages to prepare lysates is effective for alltypes of bacteria and allows the preparation of lysates to evolve withthe bacteria and avoid resistance problems common to pharmaceuticals.The use of bacteriophage lysates allows the preparation of productscapable of modulating the immune system prepared from a single bacterialstrain, or a combination of bacterial strains.

[0021] In general, methods of the present invention comprise screeningstrains of a particular bacterial family or group that does not producetoxins, for selected bacteria that exhibit the most common antigenicpattern found in the bacterial family or group. The antigenic patterncan be determined by immunogenic methods such as fluorescent antibodyidentification, FACS selection, ELISA, Western blots, SDS gelelectrophoresis or by other detection methods known to those skilled inthe art. Cultures of the selected bacteria are then infected by theintroduction of phage homologues. The combination is screened forstrains that result in quantitative lysis of bacteria in a given timeand strains that exhibit the most common antigenic pattern. Thebacterial strain and phage are then preserved, preferably bylyophylization, to maintain stability. The stability of the bacterialstrains and phages are determined through stability assays, repeatedcultures and subcultures on media. The bacterial strains and phages arealso checked for antigenic infection and reproducibility of antigenicpatterns of lysates. At least one, preferably two or more, of theresultant lysates are combined to yield an immunomodulator composition.Therefore, the resulting lysate from the lysis can be quantitatively andqualitatively analyzed by SDS gel electrophoresis.

[0022] The compositions can be used in methods for the treatment oflocal or systemic bacterial infections including, but not limited to,chronic or recurrent respiratory or ear infections, prophylacticprevention of infections and treatment of pathological conditions ofmicrobial infections such as post operational infections, bacterialpneumonia infections, sepsis, skin infections, wound infection,osteomyelitis, skin polymicrobial infections, allergies, asthma,endocarditis, arthritis, abscess, sinusitis, and acne vulgaris.Additionally, bacteriophage lysates can be used in methods for immunestimulation such as vaccines that boost the immune system. Thecompositions can also be administered for several months prior toplanned operations such as knee or hip replacements to boost generalimmune response, decrease recovery time, and prevent nosocomialinfections.

[0023] Methods of treatment contemplated by the present inventioncomprise administration of an effective amount of a bacterial lysatecomposition to an organism having an infection or in need of immunestimulation. An effective amount of a bacterial lysate composition canbe determined by using known amounts of such compositions, or bystarting treatment with a small dose and increasing the dose until thedesired effect is achieved. Such techniques for determining effectiveamounts are known to those skilled in the art and do not require undueexperimentation to determine. Differing routes of administrationincluding, but not limited to, oral, buccal, nasal, aerosol, topical,transdermal, injectable, slow release, controlled release,iontophoresis, sonophoresis, intravenous, intramuscular,intraperitoneal, intraspinal, intrathecal, intracerebroventricular,intraarterial, subcutaneous and intranasal routes, generally requirediffering effective amounts to achieve the desired result. For example,oral administration may require different effective amounts thantopically administered effective amounts. In general, a range of 0.0001mg/kg/day to 500 mg/kg/day provides an effective amount of a bacteriallysate composition.

[0024] Preferred methods comprise administration of the lysate three tosix weeks prior to reconstructive surgery such as, but not limited to, aknee or hip replacement. For example, the lysate is administered in adose is 0.05 cc, injected intradermally twice a week. Three days afteradministration of the initial dose, the patient is observed fortolerance of the lysate. If no local reaction is noted, the dose can beincreased to 0.1 cc up to 0.2 cc. The lysate is then administered everyother day. In the case of nasal routes of administration, preferably twoto four drops are placed in each nostril the first four days and afterfour days, the dosage is repeated every other day. The length oftreatment is generally three to four months. Multiple methods ofadministration may be combined with the length of treatment depending onthe immune status of the patient.

[0025] The present invention further comprises methods of eliciting animmune response in a host comprising administering an effective amountof an immunogenic composition. The immunogenic composition may be usedprophylactically as part of a vaccination system in which thecomposition is administered prior to infection, or in the treatment of aparticular infection. The immune response may be a humoral or acell-mediated immune response. Immunogenicity may be improved theco-administration of antigens with adjuvants. Adjuvants may act byretaining the antigen locally near the site of administrationfacilitating a slow sustained release of antigen to cells. Adjuvants canalso attract immune cells to the site of injection and stimulate suchcells to elicit immune responses. A wide range of adjuvants can aid inevoking an immune responses. These include, but are not limited to,pluronic polymers with mineral oil, Freund's complete adjuvant, lipid A,liposomes and cholera toxin subunit B or its genetically modifiedvariants.

[0026] The following specific examples of the methods and compositionsof the present invention are in no way to be seen as limiting, butmerely provide illustrative embodiments for the applications of thepresent invention. The present invention contemplates the use ofbacterial lysates for the treatment of bacterial and other microbialinfections and for stimulation of the immune system in general and otherdisease treatments or compositions are not limited by the specificexamples taught herein.

[0027]Staphylococcus aureus Infections Prevention and Treatment

[0028] Staphylococcal bacteria, and S. aureus in particular, are some ofthe more common and virulent pathogens. Some staphylococcal infectionsare characterized by intense suppuration, necrosis of local tissues andthe formation of abscesses. Staphylococcal infections are responsiblefor skin infections, such as furuncles, carbuncles and impetigo, anddeep lesions spreading from the skin into bones, joints, soft tissuesand organs. S. aureus can produce toxins leading to scalded skinsyndrome, toxic shock syndrome and staphylococcal food poisoning. It isa major cause of wound infection and a continuing cause of hospitalepidemics. S. aureus is currently resistant to many classes ofantibiotics and is currently being treated with vancomycin as a lastresort. However, strains resistant to vancomycin have already begun toappear.

[0029]S. aureus has many virulence factors that act against native oradaptive immune responses. While not wishing to be bound by anyparticular theory, it is currently believed that part of the innateresponse against S. aureus comprises the Toll-like receptor (TLR)family. TLRs are transmembrane receptors linking the pattern recognitionsystem of pathogens with intracellular signaling. In humans, the TLRfamily is represented by TLR (TLR 1-10), several proteins related to TLR(RP105, Nod1, Nod2), as well as lipopolysaccharide (LPS) receptor(CD14). Several TLRs appear to have a specific function in the patternrecognition system. For example, TLR-2 is believed to activate cells inresponse to gram-positive cell-wall components (peptidoglycan). TLR-4 isimportant (in complex with CD14) for recognition of LPS (gram-negativebacteria). TLR-9 recognizes CpG motif of bacterial DNA. The activationof the innate immune system induces early inflammatory reaction mediatedby monocytes, neutrophils, and endothelial cells. The response can occurwith or without participation of the adaptive immune system, leading toleukocyte recruitment, production of pro-inflammatory cytokines(TNF-alpha, aIL-1 beta), reactive oxygen products, and effectorcytokines (e.g., IL-12) acting on T cells, antigen presenting cells, andby a cascade of cytokines indirectly on B cells.

[0030] Mice deficient in TLR-2 or a signaling molecule (MyD88) werehighly susceptible to S. aureus infection (J. Immunol. 165, 5392-5396,2000). Furthermore, wild-type mice became more resistant to acutepolymicrobial sepsis after treatment with CpG DNA (J. Immunol. 165,4537-4543, 2000) that is currently believed to act via TLR-9. TLR-2 islikely to play an important role in human S. aureus infection asevidenced by detection of TLR-2 polymorphism associated with S. aureusseptic shock (Infect. Immun. 68, 6398, 2000). Activation of TLR-2receptor with S. aureus cell wall components results in upregulation ofTh-1 pathway (TNF-alpha, interpheron gamma, IL-1, etc.). Increase ofthese pro-inflammatory cytokines activates and modulates multipleresponses of innate and adaptive immunity.

[0031]S. aureus has several mechanisms to interfere with the normalimmune response. Not only has the bacteria developed a resistanceagainst antimicrobial proteins but it can also survive insideneutrophils. In addition, various strains of S. aureus possess some ofthe battery of toxins and enzymes and other products that enhanceinfectivity and bacterial survival and proliferation. Antibodiesspecific against toxins and enzymes and other soluble productsneutralize the activities of the toxins while antibodies specificagainst cells and cellular components opsonize the targeted cells andenhance their phagocytosis.

[0032] Treatment of staphylococcal infections and S. aureus inparticular, are provided by the present invention. Selectedstaphylococcal lysate compositions comprising lysate from single ormultiple strains of staphylococcus and bacteriophages are administeredthrough various routes, including but not limited to, nasal drops andsubcutaneous routes, or a combination thereof. Lysates from differentstrains of staphylococcus or lysates that result from differentbacterial phage inoculations are preferably combined in equalproportions. Staphyloccocal lysate (SPL) has been clinically effectivein the treatment of all staphylococcal infections of adults, as well aschronic conditions in pediatrics (chronic upper respiratory diseases,bronchial asthma, chronic sinusitis, cystic fibrosis) and in dermatology(acne vulgaris).

[0033] SPL is prepared by lysis of S. aureus culture with a polyvalentbacteriophage. Most staphylococcal phages belong to the Siphoviridaefamily, e.g., phages with double-stranded linear DNA and with long,noncontractile tails. SPL is a complex of antigenic components ofribosomal, cytoplasmic, nuclear, cell wall, and membranous origin.Purified components of SPL can be used for specific stimulation ofcertain pathways including, but not limited to, peptidoglycans viaTLR-2, CpG DNA via TLR-9.

[0034] Alternatively, animals and humans can be vaccinated with SPLcompositions so that staphylococcal infections are resisted. Suchtreatment comprises administration of a SPL composition comprising lysedbacteria and phage through routes of administration comprisingsubcutaneous injections, nasal drop application and lavage ofosteomyelitis fistulas. The administration of the lysate compositionscan take place prophylacticly, prior to the occurrence of astaphylococcal infection, as well as during a staphylococcal infection.Preferred times of prophylactic administration are after the age ofthree for three to six weeks prior to potential infections situations orgenerally as part of a vaccination program.

[0035]Klebsiella pneumoniae Infection Prevention and Treatment

[0036] Another bacteria group for which the present invention iseffective is the Klebsiella bacteria, in particular, K. pneumoniae. K.pneumoniae is a nonopportunistic pathogen normally found in the gut.Outside of the gut, it is a growing source of hospital-acquiredinfections causing pneumonia and urinary tract infections. Patients withchronic respiratory diseases, diabetics, alcoholics, and people ofadvanced age, as well as those using respiratory therapy devices,intravenous and urinary catheters are particularly sensitive toacquiring infectious Klebsiella.

[0037] Classically, K. pneumoniae infection of the respiratory tractcauses a severe, rapid-onset illness that often results in destructionof areas in the lung. Even with treatment, the mortality rate due to K.pneumoniae is 50%. Infected persons generally develop high fever,chills, flu-like symptoms and a cough that produces a lot of mucous.While normal bacterial pneumonia frequently resolves withoutcomplication, K. pneumoniae frequently causes lung destruction andabscesses.

[0038] Klebsiella can also cause less serious respiratory infections,such as bronchitis, which is usually a hospital-acquired infection.Other common hospital-acquired infections caused by Klebsiella areurinary tract infections, surgical wound infections and bacteremia. Allof these infections can progress to shock and death if not treated earlyin an aggressive fashion.

[0039] Methods of treatment of Klebsiella infections and K. pneumoniaein particular, are provided by the present invention. Selectedklebsiella lysate compositions comprising single or multiple strains ofklebsiella or bacteriophages are administered through various routes,including but not limited to, nasal drops and subcutaneous routes, or acombination thereof. The lysate compositions are administered dailyuntil the desired treatment is achieved.

[0040] Alternatively, animals and humans can be vaccinated or treatedprophylactically with bacterial lysate compositions so that klebsiellainfections are resisted. Such treatment comprises administration ofklebsiella lysate compositions comprising lysates from single ormultiple strains of klebsiella and phage through routes ofadministration comprising nasal drops and subcutaneous administration attimes prior to infection with klebsiella. Preferred prophylacticadministration for humans is after the age of 3, for 3-6 weeks prior topotential infectious conditions such as hospitalizations or generally aspart of a vaccination program.

[0041]Pseudomonus aeruginosa Infection Prevention and Treatment

[0042] Another frequent hospital contaminant, P. aeruginosa is aversatile, gram-negative bacterium that grows in soil, marshes, andcoastal marine habitats, as well as on plant and animal tissues. P.aeruginosa is able to grow in aqueous solutions including distilledwater. This ability results in frequent contamination of i.v. fluids,respirators, anesthesia equipment and other hospital equipment. Peoplewith cystic fibrosis, burn victims, individuals with cancer, andpatients requiring extensive stays in intensive care units areparticularly at risk of P. aeruginosa infection. Unlike manyenvironmental bacteria, P. aeruginosa has a remarkable capacity to causedisease in susceptible hosts. It has the ability to adapt to and thrivein many ecological niches, from water and soil to plant and animaltissues. P. aeruginosa can produce a number of toxic proteins which notonly cause extensive tissue damage, but also interfere with the immunesystem's defense mechanisms. These proteins range from potent toxinsthat enter and kill host cells at or near the site of colonization todegradative enzymes that permanently disrupt the cell membranes andconnective tissues in various organs.

[0043]P. aeruginosa infections are a particular problem in individualswith cystic fibrosis. Cystic fibrosis is a common lethal genetic disease(incidence 1:2,000 Caucasian births). The disease presents with ahistory of chronic lung disease, recurrent pneumonia, cough, andbronchiectasis. The lungs of cystic fibrosis patients may be colonizedwith P. aeruginosa, particularly a special mucoid strain thatcontributes to fatal complications. S. aureus is frequently present aswell.

[0044] Treatment of pseudomonas infections and P. aeroginosa inparticular, are provided by the present invention. Selected pseudomonaslysate compositions comprising lysates from single or multiple strainsare administered through various routes, including but not limited to,nasal drops and subcutaneous routes, or a combination thereof.

[0045] Alternatively, animals and humans can be vaccinated withpseudomonas lysate compositions so that pseudomonas and pneumococcalinfections are resisted. Such treatment comprises administration ofpseudomonas lysate composition comprising lysates from bacteria andphage through routes of administration comprising nasal and subcutaneousinjection at times prior to infection with pseudomonas. Preferred timesof preventative administration are after 3 years of age, for three tosix weeks prior to the potential infectious situations such as proposedhospitalization or generally as part of a vaccination program.

[0046] Mastitis

[0047] The present invention is effective for the prevention andtreatment of mastitis, particularly mastitis in dairy cattle, though anymastitis can be treated using the present invention. Mastitis in dairycattle is an inflammation of the mammary gland in response tointramammary bacterial infection, mechanical trauma, or chemical trauma.Economic losses due to mastitis are $1.7 billion dollars a year in theU.S. alone. It is thought that contagious mastitis is primarily causedby S. aureus and Streptococcal agalactiae. Environmental mastitis can becaused by a variety of different bacteria, including, but not limitedto, K. pneumoniae, Escherichia. coli, Klebsiella oxytoca, Enterobacteraerogenes, Streptococcal uberis, Streptococcal bovis, and Streptococcaldysgalactia.

[0048] Traditional prevention of bovine mastitis involves a complexregimen of daily teat-dipping with a disinfectant solution, and mayinvolve antibiotic-containing teat dips. When infection does occur,intramammary infusion of antibiotics is indicated, however this leads toincreasingly resistant strains of bacteria. Antibiotic therapy canreduce the infection so that the milk produced is saleable, but itgenerally does not lead to complete elimination of the causativeorganism. While not wishing to be bound to any particular theory,studies on mastitis have indicated that part of the problem in treatingmastitis is that a significant number of bacteria remain viable in themammary gland within phagocytic polymorphonuclear neutrophil leukocytes(PMN). When lysis of the leukocyte occurs, the phagocytized bacteria mayprovide a renewed source of mastitis producing, for example,staphylococcal regrowth. While not wishing to be bound, it is currentlybelieved that the use of bacterial lysates allows for the creation ofmemory cells, allowing the cow to respond to remaining bacteria,whenever they might appear.

[0049] In general, bovine mastitis is treated by administering aneffective amount of a bacterial lysate to a cow. The administration maybe a prophylactic administration, in that all cattle in the herd aretreated with bacterial lysate compositions, or the administration mayoccur when infection occurs in individual cows. For example, in heifers,vaccination starts at 6 months of age. Three subcutaneous injections oflysate (5 cc each) are applied between 6 and 24 months of age.

[0050] The present invention is also useful in the treatment of skininfections in other domesticated animals including canine and felinestaphylococcal skin infection, dermatitis, and other chronic infections.Treatment methods comprise the administration of an effective amount ofa bacterial lysate to domesticated animals.

[0051] Bacterial antigens are known to trigger immunomodulatory effectsin vivo. For example, in an infection of a host by a staphylococcalorganism, other bacteria and some viruses, it is believed that astaphylococus lysate composition potentiates cell-mediated as well ashumoral immunity in animals and humans. Treatment with staphylococcuslysates elevates antigen specific as well as total humoral antibody andimmunoglobulin levels creating an effective immunoadjuvant for humoralresponses as well.

[0052] Delivery and Dosage

[0053] The methods of the present invention comprise routes ofadministration that include, but are not limited to, oral, buccal,nasal, aerosol, topical, transdermal, injectable, slow release,controlled release, iontophoresis, sonophoresis, and other deliverydevices and methods. Injectable methods include, but are not limited to,intravenous, intramuscular, intraperitoneal, intraspinal, intrathecal,intracerebroventricular, intraarterial, subcutaneous and intranasalroutes.

[0054] The compositions for treating the pathologies by the presentinvention can further include a pharmaceutically acceptable carrier orexcipient. The compositions can also include other medicinal agents,pharmaceutical agents, carriers, adjuvants diluents and otherpharmaceutical preparations known to those skilled in the art. Theseagents are known to those skilled in the art and are generally describedas being biologically inactive and can be administered to patientswithout causing deleterious interactions with the active agent.

[0055] According to the invention, at least one pharmaceuticalcomposition can be delivered by any of a variety of inhalation or nasaldevices known in the art for administration of a therapeutic agent byinhalation. Devices capable of depositing aerosolized formulations inthe sinus cavity or alveoli of a patient include metered dose inhalers,nebulizers, dry powder generators, sprayers, and the like. Other devicessuitable for directing pulmonary or nasal administration are also knownin the art.

[0056] There are a several desirable features of an inhalation devicefor administering a compound of the present invention. For example,delivery by the inhalation device is advantageously reliable,reproducible, and accurate. For pulmonary administration, at least onepharmaceutical composition is delivered in a particle size effective forreaching the lower airways of the lung or sinuses.

[0057] All such inhalation devices be used for the administration of apharmaceutical composition in an aerosol. Such aerosols may compriseeither solutions (both aqueous and non aqueous) or solid particles.Metered dose inhalers like the Ventolin® metered dose inhaler, typicallyuse a propellent gas and require actuation during inspiration. See,e.g., WO 98/35888; WO 94/16970. Dry powder inhalers like Turbuhaler®(Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros® inhaler (Dura),devices marketed by Inhale Therapeutics, and the Spinhaler® powderinhaler (Fisons), use breath-actuation of a mixed powder. See U.S. Pat.Nos. 5,458,135; 4,668,218; WO 97/25086; WO 94/08552; WO 94/06498; and EP0 237 507, each entirely expressly incorporated herein by reference.Nebulizers like AERx®, Aradigm, the Ultravent® nebulizer (Mallinckrodt),and the Acorn II® nebulizer (Marquest Medical Products), the abovereferences entirely expressly incorporated herein by reference, produceaerosols from solutions, while metered dose inhalers, dry powderinhalers, etc. generate small particle aerosols. These specific examplesof commercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of theinvention, and are not intended as limiting the scope of the invention.

[0058] Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

[0059] A spray comprising a pharmaceutical composition of the presentinvention can be produced by forcing a suspension or solution of acompound disclosed herein through a nozzle under pressure. The nozzlesize and configuration, the applied pressure, and the liquid feed ratecan be chosen to achieve the desired output and particle size. Anelectrospray can be produced, for example, by an electric field inconnection with a capillary or nozzle feed.

[0060] A pharmaceutical composition of the present invention can beadministered by a nebulizer such as a jet nebulizer or an ultrasonicnebulizer. Typically, in a jet nebulizer, a compressed air source isused to create a high-velocity air jet through an orifice. As the gasexpands beyond the nozzle, a low-pressure region is created, which drawsa solution of composition protein through a capillary tube connected toa liquid reservoir. The liquid stream from the capillary tube is shearedinto unstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of composition protein either directly or through a couplingfluid, creating an aerosol including the composition protein.

[0061] In a metered dose inhaler (MDI), a propellant, a compound of thepresent invention, and any excipients or other additives are containedin a canister as a mixture including a liquefied, compressed gas.Actuation of the metering valve releases the mixture as an aerosol.

[0062] Pharmaceutical compositions for use with a metered-dose inhalerdevice will generally include a finely divided powder containing acompound disclosed herein as a suspension in a non-aqueous medium, forexample, suspended in a propellant with the aid of a surfactant. Thepropellant can be any conventional material employed for this purposesuch as chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol and1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227(hydrofluroalkane-227), or the like. One of ordinary skill in the artwill recognize that the methods of the present invention can be achievedby pulmonary administration of a compound disclosed herein via devicesnot described herein.

[0063] Simple lysate delivery systems of the present invention comprisecapsules containing differently coated pellets of the lysate. On releasefrom the capsule, the uncoated pellets provide an initial amount of thelysate composition to the body, and the coated pellets provide thelysate composition over a period of time. Another system includeshydrogel materials with coated pills embedded in the hydrogel, such asthat taught in U.S. Pat. No. 4,659,558. The unswollen hydrogel, such asthat taught in U.S. Pat. No. 4,659,558, is swallowed and in the presenceof fluids in the stomach, swells so that the hydrogel is retained withinthe stomach. The coated pills are released as the hydrogel degrades. Thelysate may also be administered through the use of auto-injectiondevices such as those described in U.S. Pat. Nos. 5,514,097; 159,192;and 5,643,214; as well as European Patent No. 0 516 473 B1.

[0064] In controlled release systems contemplated in the presentinvention, after oral ingestion, lysates are released by diffusion anderosion throughout the gastrointestinal tract to a significant degree.Methods of the present invention for the prolongation of gastricretention time include incorporation of fatty acids to reducephysiological gastric emptying and the use of bioadhesive polymers. Suchsystems are known to those skilled in the art and comprise usingpolymers such as polycarbophyll, sodium carboxymethylcellulose,tragacanth gum, acrylates and methacrylates, modified celluloses andpolysaccharide gums.

[0065] Another delivery system that is contemplated by the presentinvention for targeting lysates to the stomach while avoiding gastricemptying is known as a hydrodynamically balanced system. This system isbased on capsules or tablets with bulk density lower than gastric fluid.Thus, the dosage form stays buoyant in the stomach. These dosage formsare comprised of 20-75% of one or more hydrocolloids, e.g.,hydroxyethylcellulose and hydroxypropylmethylcellulose.

[0066] Other types of these devices include osmotic pressurecompartments containing osmotically active salts. In the presentinvention, dissolution of these salts by the gastric fluid pumps out thelysate composition. Others are based upon a floating bilayer compressedmatrix. One of the layers is comprised of a hydrophilic polymer and acarbon dioxide generating composition. The carbon dioxide maintainsbuoyancy and the other hydrophilic layer releases the drug from thematrix. A further method for gastric lysate targeting involves anintragastric retention shape, made of polyethylene or polyethyleneblend.

[0067] The delivery systems described above may also be used in thepresent invention to target lysate compositions to the upper smallintestine. However targeting to other areas of the small intestine mayinvolve several additional systems. The low stomach pH and presence ofgastric enzymes have led to the development of enteric coating. Thiscoating protects the gastric mucosa from lysate irritation. Coating isdone with a selectively insoluble substance, and protects lysates frominactivation by gastric enzymes and/or low pH.

[0068] In summary, the present invention comprises methods ofadministration of lysate compositions for treatment of microbialinfections. Not all administration routes are efficacious for everypatient. Therefore, the present invention comprises various methods,which require differing formulations of the lysate compositions. Theformulations include those suitable for oral, rectal, ophthalmic(including intravitreal or intracameral), nasal, topical (includingbuccal and sublingual), vaginal or parenteral (including subcutaneous,transdermal, intramuscular, intravenous, intradermal, intratracheal, andepidural) administration. The formulations may conveniently be presentedin unit dosage form and may be prepared by conventional pharmaceuticaltechniques.

[0069] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Molding, in a suitable machine, a mixture of thepowdered compound moistened with an inert liquid diluent may make moldedtablets. The tablets may be optionally coated or scored and may beformulated so as to provide a slow or controlled release of the lysatetherein.

[0070] Formulations suitable for topical administration in the mouthinclude lozenges comprising the ingredients in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the lysate in a suitable liquidcarrier.

[0071] The present invention additionally comprises methods of topicaladministration. To prepare the topical composition according to thepresent invention the usual manner for preparing skin care products maybe employed. The active components are generally incorporated in adermatological acceptable carrier in conventional manner. It may bepackaged in discrete units including aerosol sprays, each containing apredetermined amount of the active ingredient, as a powder, stick, orgranules, as creams, pastes, gels, lotions, syrups, or ointments, onsponges or cotton applicators, or as a solution or a suspension in anaqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil liquid emulsion. The lysates can suitably first bedissolved or dispersed in a portion of the water or another solvent orliquid to be incorporated in the composition. The composition can alsobe in the form of a so-called “wash-off” product e.g. a bath or showergel, possibly containing a delivery system for the lysates to promoteadherence to the skin during rinsing. Most preferably the product is a“leave-on” product; a product to be applied to the skin without adeliberate rinsing step soon after its application to the skin. Suchcompositions may be prepared by any of the methods of pharmacy, but allmethods include the step of bringing into association the carrier(s)with the lysate composition. In general, the compositions are preparedby uniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Thecomposition may packaged in any suitable manner such as in a jar, abottle, tube, roll-ball, or the like, in the conventional manner.

[0072] Formulations suitable for vaginal administration may be presentedas pessaries, tamports, creams, gels, pastes, foams or sprayformulations containing in addition to the lysates such carriers as areknown in the art to be appropriate.

[0073] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described. Preferred unit dosageformulations are those containing a daily dose or unit, daily sub-dose,as herein above recited, or an appropriate fraction thereof, of theadministered ingredient.

[0074] The compounds may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. REMINGTON'S PHARMACEUTICAL SCIENCES(A. Osol ed., 16th ed. (1980)).

[0075] In a specific embodiment, the compounds disclosed herein areformulated as liposomes. Liposomes containing a compound of the presentinvention are prepared by methods known in the art. See, e.g., U.S. Pat.Nos. 5,013,556; 4,485,045; 4,544,545; WO 97/38731; Epstein et al., 82PROC. NATL. ACAD. SCI. USA 3688 (1985); and Hwang et al., 77 PROC. NATL.ACAD. SCI. USA 4030 (1980). The compounds of the present invention canalso be administered in the form of liposome delivery systems such assmall unilamellar vesicles, large unilamellar vesicles, andmultilamellar vesicles. Liposomes can be formed from a variety ofphospholipids such as cholesterol, stearylamine or phophatidylcholines.

[0076] The present invention provides stable formulations as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one compound disclosed herein in apharmaceutically acceptable formulation. Formulations in accordance withthe present invention may optionally contain at least one knownpreservative.

[0077] In addition, co-administration or sequential administration ofthe compounds of the present invention and other therapeutic agents maybe desirable, such as chemotherapeutic agents, immunosuppressive agents,cytokines, cytotoxic agents, nucleolytic compounds, radioactiveisotopes, receptors, and pro-drug activating enzymes, which may benaturally occurring or produced by recombinant methods. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active therapeutic agents simultaneously exert their biologicalactivities.

[0078] In another embodiment, the other therapeutic agent comprises acytokine. The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormones such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-α andTGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

[0079] It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents.

[0080] As used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlyindicates otherwise. Thus, for example, reference to a “compound” is areference to one or more such compounds and includes equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich this invention belongs.

[0081] All publications and patents mentioned herein are incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications, which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

[0082] It is to be understood that this invention is not limited to theparticular formulations, process steps, and materials disclosed hereinas such formulations, process steps, and materials may vary somewhat. Itis also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

[0083] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing examples. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitations.

EXAMPLES Example 1 Preparation and Use of S. aureus Lysate

[0084] Strains of S. aureus bacteria were deposited with the CzechCollection of Microorganisms (CCM) and have been assigned accession no.CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM 4997, CCM 4998.The bacteriophage used were from the group deposited with the DeutscheSammlung von Mikroorganismen und Zeilkulturen GmbH (DSM) and havingaccession nos. DSM 14614, DSM 14615, DSM 14616.

[0085] The bacteria, stored as a lyophilized culture were streaked ontotrypton agar or a similar nutrient agar medium. After 24-48 h incubationat 37° C., 50 ml trypton medium was inoculated with the bacteria grownon the agar plate. The inoculated medium was incubated at 37° C. for18-20 h and the volume added to 500 ml medium and incubated for 1-2 h(based on OD measured at 600 nm). The phage stock was then added and themixture was allowed to sit at room temperature for 12-18 h. Theresulting lysate was centrifuged for 1 h at 5,000 rpm, and filteredusing filters with a pore size of 0.22 μm. The mixture was tested forsterility using blood agar and phage titration using a 2-layer agartechnique.

Example 2 Screening of Bacterial Strains

[0086] Bacterial strains are screened for the most common antigenicpattern using immunogenic methods such as fluorescent antibodyidentification, FACS selection, ELISA, Western blots, SDS gelelectrophoresis or by other detection methods known to those skilled inthe art. Generally, the antigenic pattern is measured by SDS geleletrophoresis. The components of the SPL are separated in a gel andsilver-stained. The antigenic pattern is determined by thepresence/absence of particular bands in the gel.

[0087] Cultures of the selected bacteria were then infected by theintroduction of phage homologues. The combinations of phage and bacteriawere screened for phage strains that resulted in quantitative lysis ofbacteria in a given time and strains that exhibited the most commonantigenic pattern through the above-mentioned methods. Combinations ofbacterial and phage strains that exhibit reproducible results in lysiswere selected. Combinations of one bacterial strain and different phagesresults in different antigenic combinations which can also be utilized.The selected bacterial and phage strains were preserved bylyophylization.

Example 3 Lyophilization

[0088] Phage are prepared by lysing the host bacteria resulting in 10⁹to 10¹⁰ phage particules per ml.

[0089] 0.5 ml of the phage containing bacterial lysate are placed intubes to which 0.2 ml of skim milk has been added. The tubes are thencovered with a sterile porous cover. The tubes are lyophilized using acryodesiccator instrument such as 30P2 or EdwardsEF03 using primarydesiccation for 4 h and secondary desiccation for 6 h. The tubes arethen sealed.

[0090] Each batch of phage containing bacterial lysate is tested forviability by plating the contents of one tube on a layer of hostbacteria and obtaining the titer from two-fold dilutions. The titer iscompared to the starting titer prior to lyophilization.

Example 4 Stability Analysis

[0091] The stability of the lysate cultures is determined throughstability assays which are repeated cultures and subcultures on platesand in liquid media. The stability studies are performed to selectbacterial and phage strains that are most stable upon storage. Thecriteria measured by methods known to those skilled in the art are themeasuring of antigenic pattern by SDS gel eletrophoresis; and phagevirulence, excluding strains that become resistant to the phages usedfor infection. The cultures are also checked for reproducibility ofantigenic patterns of lysates. Standardized inoculum of bacterial cellsby the phages results in a very reproducible process of lysis. Theresulting lysate is quantitatively and qualitatively analyzed by SDS gelelectrophoresis.

Example 5 Preparation of Immunomodulators

[0092] Biological assays (e.g. blastic transformation, major antigenicproteins detected by Western blots, immunomodulation, etc.) are used toidentify biologically active substances in the resultant lysates. Ablastic transformation of peripheral lymphocytes is measured usingindividual fractions from SPL fractionated by chromatographictechniques. Activation markers or thymidine incorporation is used in theassay. Active fractions (those stimulating/inhibiting activation ofperipheral lymphocytes) are further fractionated and used for isolationof a desired immunomodulator. One or more of these substances are thenpurified from one or more of the resultant lystates and identified andcombinations are formed having the desired activities. Two or more ofthe resultant lysates identified are mixed together to yield the finalpolyclonal immunomodulator.

Example 6 Treatment with S. Aureus Lysates

[0093] Patients receive a subcutaneous dose 0.05 cc-0.2 cc (5 cc bottle)of the filtrate prepared as described in Example 5 into the antebrachium(forearm). The dose is dependent on the age, clinical and immunologicalstatus of the patient. In hypersensitive patients, the doses should be0.02 cc-0.03 cc. This smaller dose is divided in half and applied intoboth antebrachiums, with the same amount administered at eachrepetition. Doses are administered every 3-5 days following theresolution of any prior local reaction. The doses are graduallyincreased in non-hypersensitive patients to 0.05 cc subcutaneously,according to individual reaction of the patient. In severe cases, thedose can be increased to 1.0 cc subcutaneous per dose. If the localreaction lasts longer then 4 days, the same dose should be repeated inthe subsequent administration.

[0094] In pediatric applications, i.e. for children aged 3-10 years, thedosage is decreased by 50% from the above regime, or may be administeredaccording to individualized reactions.

[0095] The length of the treatment is dependent on the clinical andimmunological status of the patient. Generally the lysates areadministered every 3-5 days for 3 months. This dosing regime can berepeated periodically as a booster.

[0096] In the case of milder infection or in pediatric indications thelysate can be administered as nasal drops. The dose is 4 drops in eachnostril daily for four days and thereafter every other day. Differentroutes of administration may be combined.

Example 7 Stimulation of Metabolic Burst in Phagocytic Cells (Monocytesand Polymorphonuclear Leukocytes) in Human Blood In Vitro

[0097] Samples of blood were collected and stored with heparin. Theheparinized blood was used 2 h and 7 h after collection and the100-microliter samples were incubated with SPL and other negative (noaddition) and positive controls including a peptide, formyl-MetLeuPhe(FMLP) (physiological stimulator), phorbol-12-myristate-13-acetate (PMA;strong stimulator).

[0098] A bursatest kit (Orpegen Pharma) was used for detection ofmetabolic burst in monocytes and polymorphonuclear leukocytes,respectively. Cell sorter (FACStrak) was used to count number ofpositive cells and total cells. TABLE 1 Polymorphonuclear leukocytesMonocytes (% of positive cells) (% of positive cells) Sample 2-h sample7-h sample 2-h sample 7-h sample Negative 9.7 19.4 13.9 18.1 control SPL38.2 61.7 31.3 40.2 FMLP 13.8 26.3 15.8 18.8 PMA 99.6 99.8 98.7 99.5

[0099] Staphyloccocal lysate (SPL) induced metabolic burst in bothmonocytes and polymorphonuclear leukocytes in blood samples treated 2 hand 7 h after collection. This treatment led to an increase of2.5-3.2-fold in monocytes and 3.6-4.3-fold in polymorphonuclearleukocytes versus the control.

Example 8 Activation of Sub-Population of T Cells in Human Blood InVitro

[0100] Heparinized blood was incubated for 6 h with SPL and othernegative (no addition) and positive controls including a peptideformyl-MetLeuPhe (physiological stimulator),phorbol-12-myristate-13-acetate (PMA; strong stimulator),serum-opsonized Escherichia coli cells (E. coli-o) (gram-negativebacterium containing lipopolysaccharides), and phytohemaglutinin (PHA;T-cell stimulator). Using Fastimmune test and FACStrak with triplefluorescence, early activation (CD69+) and production of interpherongamma (IFN-g) was measured in CD4+ T cells. Isotype controls were usedto subtract background staining. TABLE 2 CD69+ (CD69+) & (IFN-g+)(IFN-g+) (% of positive (% of positive (% of positive Sample cells)cells) cells) Negative 0.56 0 0.02 control PMA 76.04 3.09 0.04 fMLP 0.850 0 PHA 76.19 2.90 0.21 E. coli-o 10.12 — 0 SPL-50 ul 14.25 0.63 0.01SPL-100 ul 17.11 0.86 0.01 SPL-150 ul 18.37 0.75 0.04

[0101] SPL induced earlier and greater activation (CD69+) of CD4+ Tcells than fMLP or E.coli-o and the levels reached about 20% of thoseinduced by mitogens PMA or PHA.

[0102] When cells producing IFN-γ were counted in the CD69+subpopulation of CD4+ T lymphocytes stimulated with SPL, the levels were30% of those stimulated with mitogens PMA or PHA.

[0103]E. coli-stimulated or fMLP-stimulated CD4+ T lymphocytes did notshow any significant production of IFN-γ in the CD-69+ sub-population.In contrast, SPL-stimulated cells showed significant production of IFN-γin the early-activated (CD69+) cells.

What is claimed is:
 1. A composition, comprising a bacterial lysatederived from the infection of at least one bacterial strain ofStaphylococcus aureus (S. aureus), Klebsiella pneumoniae (K.pneumoniae), or Pseudomonas aeruginosa (P. aeruginosa) with abacteriophage.
 2. The composition of claim 1 wherein the bacteriallysate is derived from a S. aureus bacterial strain.
 3. The compositionof claim 1 wherein the lysate is derived from a S. aureus bacterialstrain deposited with the Czech Collection of Microorganisms (CCM) underaccession number CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM4997, or CCM
 4998. 4. The composition of claim 3 wherein the lysate isderived from the S. aureus bacterial strain having accession number CCM4992.
 5. The composition of claim 1, wherein the lysate is derived frommore than one bacterial strain deposited with the CCM under accessionnumber CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM 4997, orCCM 4998
 6. The composition of claim 1, wherein the S. aureus bacterialstrain comprises S. aureus having CCM accession number CCM
 4993. 7. Thecomposition of claim 1, wherein the bacterial lysate is derived from abacteriophage deposited with the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmBH (DSM) having accession number DSM 14614, DSM14615, or DSM
 14616. 8. The composition of claim 1, further comprising apharmaceutical excipient.
 9. A S. aureus bacterial isolate depositedwith the CCM under accession number CCM 4992, CCM 4993, CCM 4994, CCM4995, CCM 4996, CCM 4997, or CCM
 4998. 10. A method of treatingpathological conditions of microbial infections in humans or animalscomprising administering to a human or animal having a pathologicalcondition a composition comprising a bacterial lysate derived from theinfection of at least one bacterial strain of S. aureus, K. pneumoniaeor P. aeruginosa with a bacteriophage.
 11. The method of claim 10,wherein the composition is administered subcutaneously, intradermally,intramuscularly, nasally, or parenterally.
 12. The method of claim 11,wherein the composition is administered nasally to the human or animal.13. The method of claim 10, wherein the pathological condition ischronic upper respiratory disease, wound infection, osteomyelitis,endocarditis, skin polymicrobial infections, bronchial asthma, chronicsinusitis, cystic fibrosis or acne vulgaris.
 14. A bacteriophage,deposited with the DSM under accession number DSM 14614, DSM 14615, orDSM
 14616. 15. A method of preventing microbial infections orpathological conditions comprising administering to a human or animal acomposition comprising a bacterial lysate derived from the infection ofat least one bacterial strain of S. aureus, K. pneumoniae or P.aeruginosa with a bacteriophage.
 16. The method of claim 15, wherein thelysate is derived from a S. aureus bacterial strain deposited with theCCM under accession number CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM4996, CCM 4997, or CCM
 4998. 17. The method of claim 15, wherein thelysate is derived from a bacteriophage having accession numbers DSM14614, DSM 14615, or DSM
 14616. 18. The method of claim 15, wherein thelysate is administered prophylactically.
 19. The method of claim 15,wherein the lysate is administered three to six weeks prior to ahospitalization.
 20. The method of claim 15, wherein the bacteriallysate is administered subcutaneously, intradermally, intramuscularly,nasally, or parenterally.